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Open AccessArticle

Highly Enantioselective Production of (R)-Halohydrins with Whole Cells of Rhodotorula rubra KCh 82 Culture

Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, Wrocław 50-375, Poland
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2014, 15(12), 22392-22404;
Received: 13 October 2014 / Revised: 24 November 2014 / Accepted: 25 November 2014 / Published: 4 December 2014
(This article belongs to the Section Biochemistry)


Biotransformation of ten α-haloacetophenones in the growing culture of the strain Rhodotorula rubra KCh 82 has been carried out. Nine of the substrates underwent an effective enantioselective reduction to the respective (R)-alcohols according to Prelog’s rule, with the exception of 2-chloro-1,2-diphenylethan-1-one that was not transformed by this strain. The expected reduction proceeded without dehalogenation, leading to the respective (R)-halohydrins in high yields. The use of this biocatalyst yielded (R)-2-bromo-1-phenyl-ethan-1-ol (enantiomeric excess (ee) = 97%) and its derivatives: 4'-Bromo- (ee = 99%); 4'-Chloro- (ee > 99%); 4'-Methoxy- (ee = 96%); 3'-Methoxy- (ee = 93%); 2'-Methoxy- (ee = 98%). There were also obtained and characterized 2,4'-dichloro-, 2,2',4'-trichloro- and 2-chloro-4'-fluoro-phenyetan-1-ol with >99% of enantiomeric excesses.
Keywords: enantiospecific reduction; β2-adrenoceptor-stimulating agent; (R)-halohydrins; Rhodotorula rubra enantiospecific reduction; β2-adrenoceptor-stimulating agent; (R)-halohydrins; Rhodotorula rubra

1. Introduction

Optically active chlorohydrins are versatile intermediate products in synthesis of biologically active compounds of high importance in the pharmaceutical industry and in agriculture [1]. The important pharmaceuticals of this kind include denopamine, isoproterenol, formoterol and salmeterol, which are β-adrenergic receptor agonists (all of them with R configuration at the carbon with an OH group). Both formoterol and salmeterol are used in treatment of chronic obstructive pulmonary disease, being highly selective β2-adrenergic receptor agonists. They are administered via inhalation [2,3,4]. Denopamine is a selective β1-adrenergic agonist and a useful drug for congestive heart failure [5,6]. Whereas, isoproterenol (known also as isoprenaline) is a non-selective drug, which stimulates both β1- and β2-adrenergic receptors. It is used mainly to prevent cardiac arrhythmia that occurs when the electrical impulses to the heart are not working properly [7,8].
The key step in synthesis of β2-adrenergic receptor agonists of desired stereochemistry is enantiospecific reduction of respective α-haloacetophenones. This may be achieved either by chemical methods [9,10,11], or by biotechnological ones. The effective enantioselectivity of the desired product was achieved either by using whole cells of biocatalysts [12,13,14,15,16] or with the help of isolated enzymes, such as dehydrogenases [1,17,18] and lipases [19]. Although in the majority of the reported studies, the halohydrins were obtained with high substrate conversions and high enantiomeric excesses, there is always a risk of side products due to reductive dehalogenation (leading to respective 1-phenylehtan-1,2-diols) and substitution (2-hydroxyacetophenone and 1-phenylehtan-1,2-diol) [20,21].
These undesired processes were observed with higher percent of conversion for α-bromoacetophenone and its derivatives than for the analogous chloroacetophenones [20]. However, in synthesis of many adrenergic receptor agonists and other relative compounds bromohydrins are more useful than chlorohydrins, because a bromine atom is a better leaving group when substituted by an amine or other nucleophiles and also bromohydrins can be easier converted into epoxides (important intermediate products in synthesis of many pharmaceuticals) [22,23].
For our study on effective reduction of selected acetophenone chloro- and bromo derivatives, we have chosen Rhodotorula rubra KCh 82 strain, because of its known and described earlier ability to reduce low-molecular-weight ketones [24,25,26] and relatively low reversibility of this process [25].
The strains of this species are described in literature as expansive ones, characterized by high growth potential (even on low-cost culture media) and high production of carotenoids (a typical feature of this species is red color of cells) [27]. They are also reported as biocatalysts that possess S-specific dehydrogenases towards aliphatic-aromatic ketones [28,29,30] and ketophosphonates [31,32]. This microorganism is also capable of enantioselective hydrolysis of epoxides to lactones with high enantimeric excesses [33].

2. Results and Discussion

We performed reduction of ten substrates (mostly halogen derivatives) in the culture of the tested strain of Rhodotorula rubra KCh 82. It was only 2-chloro-1,2-diphenylethan-1-one that did not undergo biotransformation in the culture of this strain. The other substrates underwent an effective enantioselective reduction to respective (R)-alcohols, according to Prelog’s rule (Table 1).
The strains of the species R. rubra have already been employed for the enantioselective reduction of α-bromoacetophenone (1a) [34]. The process led to (R)-2-bromo-1-phenyl-ethan-1-ol (2a) with 61% enantiomeric excess and 70% yield. When an anionic surfactant was used (sodium lauryl sulfate) and the transformation was run under argon, the ee increased to 95% and the yield to 90%. Under the same conditions bioreductions of six other α-bromoacetophenones (3'-chloro, methyl, and benzyloxy derivatives) were carried out [34].
Table 1. Biotransformations of α-haloderivatives of acetophenone in the culture of R. rubra strain.
Table 1. Biotransformations of α-haloderivatives of acetophenone in the culture of R. rubra strain.
ReactionProductTime (Days)Conversion (%) aee (%) aConfig.
Ijms 15 22392 i0012a1>9988R
Ijms 15 22392 i0022b1>9999R
Ijms 15 22392 i0032c185>99R
Ijms 15 22392 i0042d19796R
Ijms 15 22392 i0052e1>9991R
Ijms 15 22392 i0062f1>9998R
Ijms 15 22392 i0072g1>9998R
Ijms 15 22392 i0082h1>9993R
Ijms 15 22392 i0092i1>9993R
a Conversion and enantiomeric excesses were determined by GC analysis using chiral columns; and Config.: Configuration.
Using lyophilized cells of Rhodotorula sp. LSL suspended in water led to compound 2a with ee > 99%, whereas adjusting the acidity of the solution to pH = 4.3 gave a product with >99% conversion [12]. The authors of this paper also described enantioselective reduction of five other α-halogen derivatives of acetophenone (including α-chloroacetophenone and its 4'-chloro and nitro derivatives, and also 3'-nitro and 4'-methoxy derivatives of α-bromoacetophenone). Therefore, taking into account all this information, we conclude that our results reported here make a good contribution to the widespread research on effective methods of receiving (R)-2-bromo-1-phenyl-ethan-1-ols. Additionally, they broaden the substrate spectrum of R. rubra strains and biocatalysis in general.
(R)-(−)-2-Bromo-1-(4'-bromophenyl)-ethan-1-ol (2b) was obtained in the culture of Rhodotorula rubra KCh 82 with >99% of substrate conversion and 99% of enantiomeric excess. For this substrate no changes in the conversion and enantiomeric excess were noticed during the biotransformation process (Table 1). It is known from literature that compound 2b can be received with 51% yield and 94% ee by enantioselective esterification of racemic compound 2b using lipase from Pseudomonas fluorescens [35]. There were reported also chemical methods of enantioselective reduction of 2-bromo-4'-bromoacetophenone (1b) to (S)-(+)-2b (89% yield and ee = 96%) using boron compounds modified with chiral phosphoric acid derivatives [36] or using BH3 modified with S-proline derivatives (99% yield and ee = 46%) [37]. It is also known that compound 2b was obtained by reduction of the respective ketone in the cultures of five Yarrowia lipolytica strains [38]. In the culture of the strain Y. lipolytica ATCC 32-338A after three days of incubation with the substrate (R)-alcohol 2b was received with both conversion and enantiomeric excess of over 99% (after one day the conversion was below 65%).
Both (S)-(+)- as well as (R)-(−)-2-bromo-1-(4'-chlorophenyl)-ethan-1-ol (2c) can be obtained by chemical methods [36,39]. In literature there are twenty microbial strains described that can reduce 2-bromo-4'-chloroacetophenone (1c) to compound 2c [38,40]. Among them, the highest substrate conversion (89%) was noted in the culture of Candida magnoliae IFO11, however, the enantiomeric excess of the alcohol obtained was only 68%. The enantiomeric excesses of over 95% were obtained in the cultures of seven biocatalysts and among them the highest substrate conversion (67%) was observed for Rhodotorula glutinis var. dairenensis IFO415 [40]. In the culture of the strain tested by us, Rhodotorula rubra KCh 82, we observed a drop in enantiomeric excess of product (2c) during biotransformation process. After one day of the reaction this alcohol was formed with ee = 99% (conversion 85%), however, during biotransformation time the conversion increased, but the enantiomeric excess decreased (Figure 1A). After six days of incubation the conversion reached >99%, but the ee dropped to 94%. Such a course of the biotransformation is the result of the enantioselective reduction of the ketone to the R-alcohol 2c, and then even more enantioselective oxidation of the R-alcohol.
The opposite change in enantioselectivity of the reduction was observed during incubation of 2-chloro-4'-chloroacetophenone (1d) in the culture of the tested strain. In this case after one day of biotransformation (R)-(−)-2-chloro-1-(4'-chlorophenyl)-ethan-1-ol (2d) was observed with ee = 96% and conversion = 97%. Both the conversion and enantiomeric excess increased with biotransformation time (>99% after nine days, Table 1). It is also possible to obtain compound (R)-2d by enzymatic methods, using commercially available keto reductases, but only with the help of coenzyme NADPH [41].
Figure 1. Time dependence of transformation of: (A) 2-bromo-4'-chloroacetophenone (1c) and (B) 2,2',4'-trichloroacetophenone (1e) in the culture of Rhodotorula rubra KCh 82.
Figure 1. Time dependence of transformation of: (A) 2-bromo-4'-chloroacetophenone (1c) and (B) 2,2',4'-trichloroacetophenone (1e) in the culture of Rhodotorula rubra KCh 82.
Ijms 15 22392 g001
Whereas, (S)-2d (ee = 96.6%) was reported to be obtained by microbiological methods from substrate 1d, using the strain Geotrichum sp. 38, though in low yield (42%) [42]. The R-isomer of alcohol 2d can be obtained also in the culture of Yarrowia lipolytica ATCC 32-338A, however, with rather low conversion (27%) and ee = 67%, after nine days of biotransformation [38]. The most effective was the process catalyzed by lyophilized cells of Rhodotorula sp. LSL suspended in water solution (pH = 4.3), conducted under argon, which gave >99% of both conversion and ee [12].
2-Chloro-1-(2',4'-dichlorophenyl)-ethan-1-ol (2e) was obtained in 97%–99% yield and with 90%–92% of ee by chemical synthesis, using triethylamine and chiral ruthenium complexes [43]. The authors of that work did not establish the absolute configuration of the product. There are also reports on reduction of 1e to 2e in the cultures of the strains of Yarrowia lipolytica. The most effective was the strain Y. lipolytica A50, which gave product 2e with >99% of conversion and ee = 48% after nine days of the reaction [38]. In the culture of the strain Rhodotorula rubra KCh 82 described here the (R)-alcohol 2e was received with >99% of conversion after one day of the transformation (ee = 91%). Extending the reaction time to six days gave pure (R)-(−)-alcohol (2e) as a single reaction product (Figure 1B).
Similar enantioselectivity was observed also for biotransformation of 2-chloro-4'-fluoroacetophenone (1f) in the culture of the tested R. rubra KCh 82 strain. The respective (R)-alcohol 2f with ee > 99% was obtained after six days of biotransformation (Table 2). In literature it has been reported that (R)-(−)-2-chloro-1-(4'-fluorophenyl)-ethan-1-ol (2f) (ee = 99%) was received by enzymatic reduction of compound 1f using alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae yeasts in the presence of coenzyme NADPH [44]. Product 2f was also obtained in biotransformation of 1f in the culture of Y. lipolytica ATCC 32-338A, however, with low conversion (12%) and ee = 48% [38]. S- and R-enantiomer of compound 2f were also received by kinetic separation with the use of lipase (Amano PS-C). This enzyme afforded (R)-2f with 53% yield and ee = 85%, along with the acetate of alcohol (S)-2f in 47% yield and ee = 95% [45].
Three methoxy derivatives of α-bromoacetophenone (1gi) were effectively reduced by Rhodotorula rubra KCh 82 (after one day of transformation the substrates were fully consumed) and we observed that the enantiomeric excesses of the respective alcohols underwent small changes during biotransformation time (Table 1). To the best of our knowledge there have been no reports on microbial reduction of 2'-methoxy and 3'-methoxy α-bromoacetophenone (1g and 1h), so far, (S)-(+)-2-bromo-1-(2'-methoxyphenylo)-ethan-1-ol (2g) can be synthesized with 99% of conversion and ee = 86% by reduction of 2-bromo-2'-methoxyacetophenone (1g) with lithium borohydride modified with chiral boron compound [46]. Racemic 2-bromo-1-(3'-methoxyphenyl)-ethan-1-ol (2h) was obtained by reduction of 2-bromo-3'-methoxyacetophenone (1h) with water/dioxane NaBH4 solution [47].
After one day of incubation of 2-bromo-4'-methoxyacetophenone (1i) in the culture of the tested R. rubra KCh 82 strain we observed the desired R-alcohol 2i with the enantiomeric excess of 93%. Due to the activity of dehydrogenases of this biocatalyst the ee of the product increased to 96% during biotransformation time. In literature it is known a synthetic method of receiving 2-bromo-1-(4'-methoxyphenyl)-ethan-1-ol (2i) by enantioselective reduction of 1i with chiral ruthenium compounds [48]. The product was obtained with 82% of conversion and 98% of enantiomeric excess (no data about the configuration, though). In this method the products of dehalogenation: compound 1i and 2i were also observed, as 18% altogether of the reaction mixture content. It is possible to obtain (R)-alcohol 2i with the enantiomeric excess of 77%–87% by enantioselective esterification of racemic 2i with the help of lipases from Pseudomonas sp. (PS-C) and Pseudomonas fluorescens [45,49]. The same alcohol (R)-2i was also obtained with >99% of both conversion and the ee using lyophilized cells of Rhodotorula sp. LSL suspended in aqueous solution of pH = 4.3 [12].

3. Experimental Section

3.1. Materials

All substrates were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Rhodotorula rubra KCh 82 strain was obtained from the Department of Chemistry of Wrocław University of Environmental and Life Sciences (Wrocław, Poland). The strain was cultivated on a Sabouraud agar consisting of aminobac (5 g), peptone K (5 g), glucose (40 g) and agar (15 g) dissolved in 1 L of distilled water, at 25 °C and pH 6.5 and stored in a refrigerator at 4 °C.

3.2. Analytical Methods

The course of biotransformation was controlled by means of Thin Layer Chromatography (TLC). Analytical TLC was carried out on silica gel G 60 F254 plates (Merck, Darmstadt, Germany). Chromatograms were developed using hexane/acetone mixture (3:1 v/v) as the eluent. Compounds were detected by spraying the plates with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in 10% H2SO4. The products were separated by column chromatography using silica gel (SiO2, Kieselgel 60, 230–400 mesh, 40–63 μm, Merck) and hexane/acetone mixture (3:1, v/v) as the developing system. Composition of biotransformation mixtures was established by gas chromatography (GC) on Agilent Technologies 7890 A GC instrument (Santa Clara, CA, USA), fitted with a flame ionization detector (FID) and a chiral column Chirasil-Dex CB (Agilent) 25 m × 0.25 mm × 0.25 μm film thickness. Temperature of injector: 200 °C; temperature of detector: 250 °C was the same for all compounds. To determinate the composition and enantiomeric excesses of product mixtures the following temperature programs were used (Table 2).
Reference samples of the racemic alcohols were prepared by reducing the ketones with sodium borohydride in methanol. NMR spectra were recorded on a DRX 600 MHz Bruker spectrometer (Bruker, Billerica, MA, USA) and measured in CDCl3. Optical rotations were measured with an Autopol IV automatic polarimeter (Rudolph, Hackettstown, NJ, USA). Absolute configurations of the products were determined by comparison of their optical rotation values with literature data.
Table 2. Temperature programs (°C) used for gas chromatography.
Table 2. Temperature programs (°C) used for gas chromatography.
Compound NumberStarting T (°C) 1 minGradient (°C·min−1)T (°C) 0 minGradient (°C·min−1)Final T (°C) 5 minRt of S-Isomer (min)Rt of R-Isomer (min)
T: Temperature.

3.3. Screening Procedure

Erlenmeyer flasks (300 mL), each containing 100 mL of the medium consisting of 3 g glucose and 1 g aminobac dissolved in water, were inoculated with a suspension of microorganisms and then incubated for 3–7 days at 25 °C on a rotary shaker (190 rpm). After full growth of the culture 20 mg of a substrate dissolved in 1 mL of acetone was added. After 1, 3, 6, 9 days of incubation under the above conditions, portions of 10 mL of the transformation mixture were taken out and extracted with CHCl3 (3 × 10 mL). The extracts were dried over MgSO4, concentrated in vacuo and analyzed by GC. All the experiments were repeated three times.

3.4. Preparative Biotransformation

The same transformations were performed on the preparative scale in 2000 mL flasks, each containing 500 mL of the cultivation medium. The cultures were incubated under the same conditions and then 200 mg of substrates dissolved in 10 mL of acetone were added to the grown cultures. After incubation the mixtures were extracted with CHCl3 (3 × 300 mL), dried (MgSO4) and concentrated in vacuo. The transformation products were separated by column chromatography and analyzed (TLC, GC, and also confirmed by 1H NMR).

3.5. Spectral Data of Isolated Metabolites

(R)-(−)-2-Bromo-1-phenylethan-1-ol (2a): A three-day transformation of substrate 1a (100 mg) in the culture R. rubra KCh 82 yielded 91 mg of compound 2a: [ α ] D 25 = −30.1° (c = 1.32 CHCl3) (97% ee), ([49], [ α ] D 25 = −33°, 93% ee); 1H NMR (CDCl3) δ: 2.68 (s, 1H, –OH), 3.59 (dd, 1H, J = 10.5, 9.0 Hz, one of –CH2–), 3.68 (dd, 1H, J = 10.5, 3.3 Hz, one of –CH2–), 4.96 (dd, 1H, J = 9.0, 3.3 Hz, –CHOH–), and 7.35–7.45 (m, 5H, H–Ar).
(R)-(−)-2-Bromo-1-(4'-bromophenyl)-ethan-1-ol (2b): A one-day transformation of substrate 1b (100 mg) in the culture R. rubra KCh 82 yielded 93 mg of compound 2b: [ α ] D 25 = −41.6° (c = 14.1 CHCl3) (99% ee), ([35], [ α ] D 25 = +56.6°, 95% ee); 1H NMR (CDCl3) δ: 2.72 (s, 1H, –OH), 3.52 (dd, 1H, J = 10.5, 8.8 Hz, one of –CH2–), 3.62 (dd, 1H, J = 10.5, 3.4 Hz, one of –CH2–), 4.91 (dd, 1H, J = 8.8, 3.4 Hz, –CHOH–), 7.28–7.38 (m, 2H, H-3' and H-5'), and 7.53–7.59 (m, 2H, H-2' and H-6').
(R)-(−)-2-Bromo-1-(4'-chlorophenyl)-ethan-1-ol (2c): A three-day transformation of substrate 1c (100 mg) in the culture R. rubra KCh 82 yielded 79 mg of compound 2c: [ α ] D 25 = −36.5° (c = 3.94 CHCl3) (98% ee), ([50], [ α ] D 25 = +39.0°, 89% ee); 1H NMR (CDCl3) δ 2.80 (s, 1H, –OH), 3.52 (dd, 1H, J = 10.5, 8.8 Hz, one of –CH2–), 3.61 (dd, 1H, J = 10.5, 3.4 Hz, one of –CH2–), 4.92 (dd, 1H, J = 8.8, 3.4 Hz, –CHOH–), and 7.36–7.48 (m, 4H, H–Ar).
(R)-(−)-2-Chloro-1-(4'-chlorophenyl)-ethan-1-ol (2d): A three-day transformation of substrate 1d (100 mg) in the culture R. rubra KCh 82 yielded 93 mg of compound 2d: [ α ] D 25 = −47.1° (c = 1.47 CHCl3) (99% ee), 1H NMR (CDCl3) δ 2.71 (s, 1H, –OH), 3.63 (dd, 1H, J = 11.3, 8.6 Hz, one of –CH2–), 3.73 (dd, 1H, J = 11.3, 3.3 Hz, one of –CH2–), 4.90 (dd, 1H, J = 8.6, 3.3 Hz –CHOH–), and 7.36–7.59 (m, 4H, H–Ar).
(R)-(−)-2-Chloro-1-(2',4'-dichlorophenyl)-ethan-1-ol (2e): A one-day transformation of substrate 1e (100 mg) in the culture R. rubra KCh 82 yielded 93 mg of compound 2e: [ α ] D 25 = −31.9° (c = 2.55 CHCl3) (99% ee), ([1], [ α ] D 25 = −52.8°, 99% ee); 1H NMR (CDCl3) δ: 2,84 (s, 1H, –OH), 3.55 (dd, 1H, J = 11.3, 8.5 Hz, one of –CH2–), 3.90 (dd, 1H, J = 11.3, 2.8 Hz, one of –CH2–), 5.29 (dd, 1H, J = 8.5, 2.8 Hz, –CHOH–), 7.34 (dd, 1H, J = 8.4, 2.1 Hz, H-5'), 7.40 (d, H1, J = 2.1 Hz, H-6'), and 7.60 (d, 1H, J = 8.4 Hz, H-3').
(R)-(−)-2-Chloro-1-(4'-fluorophenyl)-ethan-1-ol (2f): A one-day transformation of substrate 1f (100 mg) in the culture R. rubra KCh 82 yielded 88 mg of compound 2f: [ α ] D 25 = −38.6° (c = 1.10 CHCl3), (98% ee) ([51], [ α ] D 25 = +51.1°, 99% ee), 1H NMR (CDCl3) δ 2.62 (s, 1H, –OH), 3.65 (dd, 1H, J = 11.3, 8.8 Hz, one of –CH2–), 3.74 (dd, 1H, J = 11.3, 3.5 one of Hz, –CH2–) 4.74 (dd, 1H, J = 8.8, 3.5 Hz, –CHOH–), 7.09 (m, 2H Wh = 23.3 Hz, H-3' and H-5'), and 7.40 (m, 2H, Wh = 19.6 Hz, H-2' and H-6').
(R)-(−)-2-Bromo-1-(2'-methoxyphenyl)-ethan-1-ol (2g): A one-day transformation of substrate 1g (100 mg) in the culture R. rubra KCh 82 yielded 88 mg of compound 2g: = [ α ] D 25 15.3°, (c = 1.26 CHCl3), (98% ee), 1H NMR (CDCl3) δ: 2.93 (s, 1H, –OH), 3.56 (dd, 1H, J = 10.2, 8.6 Hz, one of –CH2–), 3.79 (dd, 1H, J = 10.2, 3.6 Hz, one of –CH2–), 3.89 (s, 3H, –OCH3), 5.18 (dd, 1H, J = 8.6, 3.6 Hz –CHOH–), 6.92 (d, 1H, J = 8.2 Hz, H-3'), 7.02 (td, 1H, J = 7.5, 0.7 Hz, H-5'), 7.33 (td, 1H, J = 8.2, 1.6 Hz, H-4'), and 7.47 (dd, 1H, J = 7.5, 1.6 Hz, H-6').
(R)-(−)-2-Bromo-1-(3'-methoxyphenyl)-ethan-1-ol (2h): A one-day transformation of substrate 1h (100 mg) in the culture R. rubra KCh 82 yielded 73 mg of compound 2h: [ α ] D 25 = −12.8° (c = 1.36 CHCl3) (94% ee); 1H NMR (CDCl3) δ: 2.52 (s, 1H, –OH), 3.56 (dd, 1H, J = 10.5, 9.0 Hz, one of –CH2–), 3.66 (dd, 1H, J = 10.5, 3.3 Hz, one of –CH2–), 3.84 (s, 3H, –OCH3), 4.93 (dd, 1H, J = 9.0, 3.3 Hz, –CHOH–), 6.89 (ddd, 1H, J = 8.3, 2.4, 1.1 Hz, H-4'), 6.98–7.09 (m, 2H, H-2' and H-6'), and 7.32 (t, 1H, J = 8.3 Hz, H-5').
(R)-(−)-2-Bromo-1-(4'-methoxyphenyl)-ethan-1-ol (2i): A one-day transformation of substrate 1i (100 mg) in the culture R. rubra KCh 82 yielded 76 mg of compound 2i: [ α ] D 25 = −20.3° (c = 1.96 CHCl3), (96% ee); ([52], [ α ] D 25 = +19.6°, 96% ee for enatiomer S); 1H NMR (CDCl3) δ: 2.61 (s, 1H, –OH), 3.56 (dd, 1H, J = 10.4, 9.0 Hz, one of –CH2–), 3.63 (dd, 1H, J = 10.4, 3.4 Hz, one of –CH2–), 3.84 (s, 3H, –OCH3), 4.91 (dd, 1H, J = 9.0, 3.4 Hz –CHOH–), 6.92–6.95 (m, 2H, H-3' and H-5'), and 7.33–7.36 (m, 2H, Hz, H-2' and H-6').

4. Conclusions

The presented results revealed that the strain Rhodotorula rubra KCh 82 used for the biotransformations of ten 2-halogen derivatives of acetophenone showed high R-selectivity of the reduction of the substrates. With the help of this biocatalyst, we obtained nine (R)‑halohydrins with high both yields and enantiomeric excesses, which were fully characterized spectroscopically and are potential synthons for synthesis of β-adrenergic receptor agonists. It should be underlined that (R)-halohydrins were single products of these reactions. No side products were observed.


This research was financially supported supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018.

Author Contributions

Tomasz Janeczko performed experiments; Tomasz Janeczko and Edyta Kostrzewa-Susłow designed and interpreted experiments; Tomasz Janeczko wrote the manuscript; Monika Dymarska performed microbiological examination and edited the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Zhu, D.; Hyatt, B.A.; Hua, L. Enzymatic hydrogen transfer reduction of α-chloro aromatic ketones catalyzedby a hyperthermophilic alcohol dehydrogenase. J. Mol. Catal. B-Enzym. 2009, 56, 272–276. [Google Scholar]
  2. Palmqvist, M.; Ibsen, T.; Mellén, A.; Lötvall, J. Comparison of the relative efficacy of formoterol and salmeterol in asthmatic patients. Am. J. Respir. Crit. Care.Med. 1999, 160, 244–249. [Google Scholar]
  3. Frois, C.; Wu, E.Q.; Ray, S.; Colice, G.L. Inhaled corticosteroids or long-acting β-agonists alone or in fixed-dose combinations in asthma treatment: A systematic review of fluticasone/budesonide and formoterol/salmeterol. Clin. Ther. 2009, 31, 2779–2803. [Google Scholar]
  4. Cazzola, M.; Materas, M.G.; Santangelo, G.; Vinciguerra, A.; Rossi, F.; D’Amato, G. Salmeterol and formoterol in partially reversible severe chronic obstructive pulmonary disease: A dose-response study. Respir. Med. 1995, 89, 357–362. [Google Scholar]
  5. Lee, D.-M.; Lee, J.-C.; Jeong, N.; Lee, K.-I. Asymmetric transfer hydrogenation of 2-tosyloxy-1-(4-hydroxyphenyl)ethanone derivatives: Synthesis of (R)-tembamide, (R)-aegeline, (R)-octopamine, and (R)-denopamine. Tetrahedron-Asymmetry 2007, 18, 2662–2667. [Google Scholar]
  6. Yadav, J.S.; Nanda, S.; Ready, P.T.; Rao, A.B. Stereoselective synthesis of (R)-(−)-denopamine, (R)-(−)-tembamide and (R)-(−)-aegeline via asymmetric reduction of azidoketones by Daucus carota in aqueous medium. Tetrahedron-Asymmetry 2001, 12, 3381–3385. [Google Scholar]
  7. Abrass, I.B.; Davis, J.L.; Scarpace, P.J. Isoproterenol responsiveness and myocardial β-adrenergic receptors in young and old rats. J. Gerontol. 1982, 37, 156–160. [Google Scholar]
  8. Zhang, G.-X.; Kimura, S.; Nishiyama, A.; Shokoji, T.; Rahman, M.; Yao, L.; Nagai, Y.; Fujisawa, Y.; Miyatake, A.; Abe, Y. Cardiac oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc. Res. 2005, 65, 230–238. [Google Scholar]
  9. Morris, D.J.; Hayes, A.M.; Wills, M. The “Reverse-Tethered” ruthenium (II) catalyst for asymmetric transfer hydrogenation: Further applications. J. Org. Chem. 2006, 71, 7035–7044. [Google Scholar]
  10. Ohkuma, T.; Tsutsumi, K.; Utsumi, N.; Arai, N.; Noyori, R.; Murata, K. Asymmetric hydrogenation of α-chloro aromatic ketones catalyzed by η6-arene/TsDPEN−ruthenium(II) complexes. Org. Lett. 2007, 9, 255–257. [Google Scholar]
  11. Wu, X.-F.; Min, C.; Nyamzundui, E.; Zhou, H.-B.; Dong, C. A novel C3-symmetric prolinol-squaramide catalyst for the asymmetric reduction of ketones by borane. Tetrahedron-Asymmetry 2011, 22, 1640–1643. [Google Scholar]
  12. Aguirre-Pranzoni, C.; Bisogno, F.R.; Orden, A.A.; Kurina-Sanz, M. Lyophilized Rhodotorula yeast as all-in-one redox biocatalyst: Access to enantiopure building blocks by simple chemoenzymatic one-pot procedures. J. Mol. Catal. B-Enzym 2014. [Google Scholar] [CrossRef]
  13. Fardelone, L.C.; Rodrigues, J.A.R.; Moran, P.J.S. Chiral pharmaceutical intermediaries obtained by reduction of 2-halo-1-(4-substituted phenyl)-ethanones mediated by Geotrichum candidum CCT 1205 and Rhodotorula glutinis CCT 2182. Enzym. Res. 2011, 2011, 976368. [Google Scholar]
  14. Taketomi, S.; Asano, M.; Higashi, T.; Shoji, M.; Sugai, T. Chemo-enzymatic route for (R)-terbutaline hydrochloride based on microbial asymmetric reduction of a substituted α-chloroacetophenone derivative. J. Mol. Catal. B-Enzym. 2012, 84, 83–88. [Google Scholar]
  15. Tokoshima, D.; Hanaya, K.; Shoji, M.; Sugai, T. Whole-cell yeast-mediated preparation of (R)-2-chloro-1-(3-nitrophenyl)ethanol as a synthetic precursor for (R)-phenylephrine. J. Mol. Catal. B-Enzym. 2013, 97, 95–99. [Google Scholar]
  16. Asami, K.; Machida, T.; Jung, S.; Hanaya, K.; Shoji, M.; Sugai, T. Synthesis of (R)-bambuterol based on asymmetric reduction of 1-[3,5-bis(dimethylcarbamoyloxy)phenyl]-2-chloroethanone with incubated whole cells of Williopsis californica JCM 3600. J. Mol. Catal. B-Enzym. 2013, 97, 106–109. [Google Scholar]
  17. Ren, J.; Dong, W.; Yu, B.; Wu, Q.; Zhu, D. Synthesis of optically active a-bromohydrins via reduction of α-bromoacetophenone analogues catalyzed by an isolated carbonyl reductase. Tetrahedron-Asymmetry 2012, 23, 497–500. [Google Scholar]
  18. Rowan, A.S.; Moody, T.S.; Howard, R.M.; Underwood, T.J.; Miskelly, I.R.; He, Y.; Wang, B. Preparative access to medicinal chemistry related chiral alcohols using carbonyl reductase technology. Tetrahedron-Asymmetry 2013, 24, 1369–1381. [Google Scholar]
  19. Xia, S.W.; Lin, H.; Chen, Y.Z. Preparation of (R)-2-chloro-1-(m-chlorophenyl)ethanol by Lipozyme TL IM-catalyzed second resolution. Chin. Chem. Lett. 2012, 23, 289–292. [Google Scholar]
  20. Rocha, L.C.; Ferreira, H.V.; Pimenta, E.F.; Souza Berlinck, R.G.; Oliveira Rezende, M.O.; Landgraf, M.D.; Regali Seleghim, M.H.; Durães Sette, L.; Meleiro Porto, A.L. Biotransformation of α-bromoacetophenones by the marine fungus Aspergillus sydowii. Mar. Biotechnol. 2010, 12, 552–557. [Google Scholar]
  21. Utsukihara, T.; Okada, S.; Kato, N.; Horiuchi, C.A. Biotransformation of α-bromo and α,α'-dibromo alkanone to α-hydroxyketone and α-diketone by Spirulina platensis. J. Mol. Catal. B-Enzym. 2007, 45, 68–72. [Google Scholar]
  22. Bisogno, F.R.; Cuetos, A.; Orden, A.A.; Kurina-Sanz, M.; Lavandera, I.; Gotor, V. Chemo- and stereodivergent preparation of terminal epoxides and bromohydrins through one-pot biocatalysed reactions: Access to enantiopure five- and six-membered N-heterocycles. Adv. Synth. Catal. 2010, 352, 1657–1661. [Google Scholar]
  23. Gopishetty, B.; Gogoi, S.; Dutta, A.K. An improved asymmetric synthetic route to a novel triple uptake inhibitor antidepressant (2S,4R,5R)-2-benzhydryl-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-142). Tetrahedron-Asymmetry 2011, 22, 1081–1086. [Google Scholar]
  24. Janeczko, T.; Gładkowski, W.; Kostrzewa-Susłowa, E. Microbial production of dihydrochalcones and its derivatives as the food sweeteners. J. Mol. Catal. B-Enzym. 2013, 98, 55–61. [Google Scholar]
  25. Janeczko, T.; Kostrzewa-Susłowa, E. Enantioselective reduction of propiophenone formed from 3-chloropropiophenone and stereoinversion of resulting alcohols in selected yeast cultures. Tetrahedron-Asymmetry 2014, 25, 1264–1269. [Google Scholar]
  26. Janeczko, T.; Dymarska, M.; Siepka, M.; Gniłka, R.; Leśniak, A.; Popłoński, J.; Kostrzewa-Susłow, E. Enantioselective reduction of flavanone and oxidation of cis- and trans-flavan-4-ol by selected yeast cultures. J. Mol. Catal. B-Enzym. 2014, 109, 47–52. [Google Scholar]
  27. Banzatto, D.; de Freita, L.A.; Mutton, M.J.R. Carotenoid production by Rhodotorula rubra cultivated in sugarcane juice, molasses, and syrup. Ciênc. Tecnol. Aliment. 2013, 33, 14–18. [Google Scholar]
  28. De Oliveira Lopes, R.; Benzaquem Ribeiro, J.; de Souza Ramos, A.; Miranda, L.S.M.; Ramos Leal, I.C.; Gomes Ferreira Leite, S.; Alves de Souza, R.O.M. Highly enantioselective bioreduction of 4-bromoacetophenone. Tetrahedron-Asymmetry 2011, 22, 1763–1766. [Google Scholar]
  29. Roy, S.; Alexandre, V.; Neuwels, M.; le Texier, L. Asymmetric bioreduction of a bulky ketone: 1-Phenyl-1-(2-phenylthiazol-5-yl)-methanone. Adv. Synth. Catal. 2001, 343, 738–743. [Google Scholar]
  30. Lorraine, K.; King, S.; Greasham, R.; Chartrain, M. Asymmetric bioreduction of a ketosulfone to the corresponding trans-hydroxysulfone by the yeast Rhodotorula rubra MY 2169. Enzym. Microb. Technol. 1996, 19, 250–255. [Google Scholar]
  31. Brzezińska-Rodak, M.; Żymańczyk-Duda, E.; Kafarski, P.; Lejczak, B. Application of fungi as biocatalysts for the reduction of diethyl 1-oxoalkylphosphonates in anhydrous hexane. Biotechnol. Prog. 2002, 18, 1287–1291. [Google Scholar]
  32. Żymańczyk-Duda, E.; Klimek-Ochab, M.; Kafarski, P.; Lejczak, B. Stereochemical control of biocatalytic asymmetric reduction of diethyl 2-oxopropylphosphonate employing yeasts. J. Organomet. Chem. 2005, 690, 2593–2596. [Google Scholar]
  33. Olejniczak, T.; Grabarczyk, M.; Wawrzeńczyk, C. Lactones 7: Enantioselective lactonization of racemic ethyl (5,5-dimethyl-2,3-epoxycyclohex-1-yl)acetate. J. Mol. Catal. B-Enzym. 2001, 11, 243–247. [Google Scholar]
  34. Goswami, A.; Bezbaruah, R.L.; Goswami, J.; Borthakur, N.; Dey, D.; Hazarika, A.K. Microbial reduction of ω-bromoacetophenones in the presence of surfactants. Tetrahedron-Asymmetry 2000, 11, 3701–3709. [Google Scholar]
  35. Hiratake, J.; Inagaki, M.; Nishioka, T.; Oda, J. Irreversible and highly enantioselective acylation of 2-halo-1-arylethanols in organic solvents catalyzed by a lipase from Pseudomonas fluorescens. J. Org. Chem. 1988, 53, 6130–6133. [Google Scholar]
  36. Basavaiah, D.; Jayapal Reddy, G.; Chandrashekar, V. A novel and effective chiral phosphoramide catalyst for the borane-mediated asymmetric reduction of prochiral a-halo ketones. Tetrahedron-Asymmetry 2001, 12, 685–689. [Google Scholar]
  37. Olivares-Romero, J.L.; Juaristi, E. Synthesis of three novel chiral diamines derived from (S)-proline and their evaluation as precursors of diazaborolidines for the catalytic borane-mediated enantioselective reduction of prochiral ketones. Tetrahedron 2008, 64, 9992–9998. [Google Scholar]
  38. Janeczko, T.; Bakowski, W.; Walczak, E.; Robak, M.; Dmochowska-Gładysz, J.; Kostrzewa-Susłow, E. Biotransformation of acetophenone and its halogen derivatives by Yarrowia lipolytica strains. Ann. Microbiol. 2014. [Google Scholar] [CrossRef]
  39. Bandini, M.; Cozzi, P.G.; Negro, L.; Umani-Ronchi, A. Enantioselective reduction of ketones with triethoxysilane catalyzed by chiral bis-oxazoline titanium complexes. Chem. Commun. 1999, 39. [Google Scholar]
  40. Kizaki, N.; Sawa, I.; Yano, M.; Yasohara, Y.; Hasegawa, J. Purification and characterization of a yeast carbonyl reductase for synthesis of optically active (R)-styrene oxide derivatives. Biosci. Biotechnol. Biochem. 2005, 69, 79–86. [Google Scholar]
  41. Zhu, D.; Mukherjee, C.; Hua, L. “Green” synthesis of important pharmaceutical building blocks: Enzymatic access to enantiomerically pure α-chloroalcohols. Tetrahedron-Asymmetry 2005, 16, 3275–3278. [Google Scholar]
  42. Wei, Z.-L.; Li, Z.-Y.; Lin, G.-Q. Anti-Prelog microbial reduction of aryl α-halomethyl or α-hydroxymethyl ketones with Geotrichum sp. 38. Tetrahedron 1998, 54, 13059–13072. [Google Scholar]
  43. Huanga, X.; Ying, J.Y. Asymmetric transfer hydrogenation over Ru–TsDPEN catalysts supported on siliceous mesocellular foam. Chem. Commun. 2007, 18, 1825–1827. [Google Scholar]
  44. Yang, Y.; Zhu, D.; Piegat, T.J.; Hua, L. Enzymatic ketone reduction: mapping the substrate profile of a short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae. Tetrahedron-Asymmetry 2007, 18, 1799–1803. [Google Scholar]
  45. Pàmies, O.; Bäckvall, J.-E. Chemoenzymatic dynamic kinetic resolution of β-halo alcohols. An efficient route to chiral epoxides. J. Org. Chem. 2002, 67, 9006–9010. [Google Scholar]
  46. Cordes, D.B.; Kwong, T.J.; Morgan, K.A.; Singaram, B. Chiral styrene oxides from α-haloacetophenones using NaBH4 and TarB-NO2, a chiral Lewis acid. Tetrahedron Lett. 2006, 47, 349–351. [Google Scholar]
  47. Perrone, R.; Berardi, F.; Leopoldo, M.; Tortorella, V. Oxygen isosteric derivatives of 3-(3-hydroxypheny1)-N-n-propylpiperidine. J. Med. Chem. 1992, 35, 3045–3049. [Google Scholar]
  48. Wang, F.; Liu, H.; Cun, L.; Zhu, J.; Deng, J.; Jiang, Y. Asymmetric transfer hydrogenation of ketones catalyzed by hydrophobic metal-amido complexes in aqueous micelles and vesicles. J. Org. Chem. 2005, 70, 9424–9429. [Google Scholar]
  49. Smith, H.E.; Fontana, L.P. Optically active amines A sector rule for the circular dichroism of the benzene chromophore. J. Org. Chem. 1991, 56, 432–435. [Google Scholar]
  50. Basavaiah, D.; Reddy, G.J.; Chandrashekar, V. A new chiral catalytic source with an N–P2O structural framework containing a proximal hydroxyl group for the borane-mediated asymmetric reduction of prochiral ketones. Tetrahedron-Asymmetry 2004, 15, 47–52. [Google Scholar]
  51. Lin, H.; Chen, Y.; Xu, X.; Xia, S.; Wang, L. Preparation of key intermediates of adrenergic receptor agonists: Highly enantioselective production of (R)-α-halohydrins with Saccharomyces cerevisiae CGMCC 2.396. J. Mol. Catal. B-Enzym. 2009, 57, 1–5. [Google Scholar]
  52. Yang, S.-D.; Shi, Y.; Sun, Z.-H.; Zhaoa, Y.-B.; Liang, Y.-M. Asymmetric borane reduction of prochiral ketones using imidazolium-tagged sulfonamide catalyst. Tetrahedron-Asymmetry 2006, 17, 1895–1900. [Google Scholar]
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